Benjamin J. Adler

Quantification of adiposity in small rodents using micro-CT

Non-invasive three-dimensional imaging of live rodents is a powerful research tool that has become critical for advances in many biomedical fields. For investigations into adipose development, obesity, or diabetes, accurate and precise techniques that quantify adiposity in vivo are critical. Because total body fat mass does not accurately predict health risks associated with the metabolic syndrome, imaging modalities should be able to stratify total adiposity into subcutaneous and visceral adiposity. Micro-computed tomography (micro-CT) acquires high-resolution images based on the physical density of the material and can readily discriminate between subcutaneous and visceral fat. Here, a micro-CT based method to image the adiposity of live rodents is described. An automated and validated algorithm to quantify the volume of discrete fat deposits from the computed tomography is available. Data indicate that scanning the abdomen provides sufficient information to estimate total body fat. Very high correlations between micro-CT determined adipose volumes and the weight of explanted fat pads demonstrate that micro-CT can accurately monitor site-specific changes in adiposity. Taken together, in vivo micro-CT is a non-invasive, highly quantitative imaging modality with greater resolution and selectivity, but potentially lower throughput, than many other methods to precisely determine total and regional adipose volumes and fat infiltration in live rodents.

Devastation of adult stem cell pools by irradiation precedes collapse of trabecular bone quality and quantity.

Stem cell depletion and compromised bone marrow resulting from radiation exposure fosters long‐term deterioration of numerous physiologic systems, with the degradation of the skeletal system ultimately increasing the risk of fractures. To study the interrelationship of damaged bone marrow cell populations with trabecular microarchitecture, 8‐ and 16‐week‐old C57BL/6 male mice were sublethally irradiated with 5 Gy of 137Cs γ‐rays, and adult stem cells residing in the bone marrow, as well as bone quantity and quality, were evaluated in the proximal tibia after 2 days, 10 days, and 8 weeks compared with age‐matched controls. Total extracted bone marrow cells in the irradiated 8‐week, young adult mice, including the hematopoietic cell niches, collapsed by 65% ± 11% after 2 days, remaining at those levels through 10 days, only recovering to age‐matched control levels by 8 weeks. As early as 10 days, double‐labeled surface was undetectable in the irradiated group, paralleled by a 41% ± 12% and 33% ± 4% decline in bone volume fraction (BV/TV) and trabecular number (Tb.N), respectively, and a 50% ± 10% increase in trabecular separation (Tb.Sp) compared with the age‐matched controls, a compromised structure that persisted to 8 weeks postirradiation. Although the overall collapse of the bone marrow population and devastation of bone quality was similar between the “young adult” and “mature” mice, the impact of irradiation—and the speed of recovery—on specific hematopoietic subpopulations was dependent on age, with the older animals slower to restore key progenitor populations. These data indicate that, independent of animal age, complications arising from irradiation extend beyond the collapse of the stem cell population and extend toward damage to key organ systems. It is reasonable to presume that accelerating the recovery of these stem cell pools will enable the prompt repair of the skeletal system and ultimately reduce the susceptibility to fractures.

Obesity-driven disruption of haematopoiesis and the bone marrow niche

Obesity markedly increases susceptibility to a range of diseases and simultaneously undermines the viability and fate selection of haematopoietic stem cells (HSCs), and thus the kinetics of leukocyte production that is critical to innate and adaptive immunity. Considering that blood cell production and the differentiation of HSCs and their progeny is orchestrated, in part, by complex interacting signals emanating from the bone marrow microenvironment, it is not surprising that conditions that disturb bone marrow structure inevitably disrupt both the numbers and lineage-fates of these key blood cell progenitors. In addition to the increased adipose burden in visceral and subcutaneous compartments, obesity causes a marked increase in the size and number of adipocytes encroaching into the bone marrow space, almost certainly disturbing HSC interactions with neighbouring cells, which include osteoblasts, osteoclasts, mesenchymal cells and endothelial cells. As the global obesity pandemic grows, the short-term and long-term consequences of increased bone marrow adiposity on HSC lineage selection and immune function remain uncertain. This Review discusses the differentiation and function of haematopoietic cell populations, the principal physicochemical components of the bone marrow niche, and how this environment influences HSCs and haematopoiesis in general. The effect of adipocytes and adiposity on HSC and progenitor cell populations is also discussed, with the goal of understanding how obesity might compromise the core haematopoietic system.

Bone structure and B-cell populations, crippled by obesity, are partially rescued by brief daily exposure to low-magnitude mechanical signals

Deterioration of the immune and skeletal systems, each of which parallel obesity, reflects a fragile interrelationship between adiposity and osteoimmunology. Using a murine model of diet‐induced obesity, this study investigated the ability of mechanical signals to protect the skeletal‐immune systems at the tissue, cellular, and molecular level. A long‐term (7 mo) high‐fat diet increased total adiposity (+62%), accelerated age‐related loss of trabecular bone (–61%), and markedly reduced B‐cell number in the marrow (–52%) and blood (–36%) compared to mice fed a regular diet. In the final 4 mo of the protocol, the application of low‐magnitude mechanical signals (0.2 g at 90 Hz, 15 min/d, 5 d/wk) restored both bone structure and B cells to those levels measured in control mice fed a regular diet. These phenotypic outcomes were achieved, in part, by reductions in osteoclastic activity and a biasing of hematopoietic stem cell differentiation toward the lymphoid B‐cell lineage and away from a myeloid fate. These results emphasize that obesity undermines both the skeletal and immune systems, yet brief exposure to mechanical signals, perhaps as a surrogate to the salutary influence of exercise, diminishes the consequences of diabetes and obesity, restoring bone structure and normalizing B‐cell populations by biasing of the fate of stem cells through mechanosensitive pathways.—Chan, M. E., Adler, B. J., Green, D. E., Rubin, C. T. Bone structure and B‐cell populations, crippled by obesity, are partially rescued by brief daily exposure to low‐magnitude mechanical signals. FASEB J. 26, 4855–4863 (2012). www.fasebj.org

High Fat Diet Rapidly Suppresses B Lymphopoiesis by Disrupting the Supportive Capacity of the Bone Marrow Niche

The bone marrow (BM) niche is the primary site of hematopoiesis, and cues from this microenvironment are critical to maintain hematopoiesis. Obesity increases lifetime susceptibility to a host of chronic diseases, and has been linked to defective leukogenesis. The pressures obesity exerts on hematopoietic tissues led us to study the effects of a high fat diet (HFD: 60% Kcal from fat) on B cell development in BM. Seven week old male C57Bl/6J mice were fed either a high fat (HFD) or regular chow (RD) diet for periods of 2 days, 1 week and 6 weeks. B-cell populations (B220+) were not altered after 2 d of HFD, within 1 w B-cell proportions were reduced by −10%, and by 6 w by −25% as compared to RD (p<0.05). BM RNA was extracted to track the expression of B-cell development markers Il-7, Ebf-1 and Pax-5. At 2 d, the expression of Il-7 and Ebf-1 were reduced by −20% (p = 0.08) and −11% (p = 0.06) whereas Pax-5 was not significantly impacted. At one week, however, the expressions of Il-7, Ebf-1, and Pax-5 in HFD mice fell by -19%, −20% and −16%, and by six weeks were further reduced to −23%, −29% and −34% as compared to RD (p<0.05 for all), a suppression paralleled by a +363% increase in adipose encroachment within the marrow space (p<0.01). Il-7 is a critical factor in the early B-cell lineage which is secreted by supportive cells in the BM niche, and is necessary for B-cell commitment. These data indicate that BM Il-7 expression, and by extension B-cell differentiation, are rapidly impaired by HFD. The trend towards suppressed expression of Il-7 following only 2 d of HFD demonstrates how susceptible the BM niche, and the cells which rely on it, are to diet, which ultimately could contribute to disease susceptibility in metabolic disorders such as obesity.

Low magnitude mechanical signals mitigate osteopenia without compromising longevity in an aged murine model of spontaneous granulosa cell ovarian cancer.

Cancer progression is often paralleled by a decline in bone mass, raising risk of fracture. Concerns persist regarding anabolic interventions for skeletal protection, as these may inadvertently exacerbate neoplastic tissue expansion. Given bones inherent mechanosensitivity, low intensity vibration (LIV), a mechanical signal that encourages osteoblastogenesis, could possibly slow cancer-associated bone loss, but this goal must be achieved without fostering disease progression. Seventy 12w female F1-SWRxSWXJ-9 mice, a strain prone to developing granulosa cell tumors, were randomized into baseline control (BC: n=10), age-matched control (AC: n=30), and LIV (n=30), which received mechanical signals (90Hz @ 0.3g) for 15m/day, 5 day/w over the course of 1 year. Survival curves for AC (10 died) and LIV (8 died) followed similar trends (p=0.62), indicating longevity was unperturbed by LIV. At 1 year, bone volume of proximal tibiae in LIV mice was 25% greater than AC (p<0.02), while bone volume of L5 vertebrae was 16% higher in LIV over AC (p<0.02). Primary lesions and peripheral metastases were apparent in both LIV and AC; however, overall tumor incidence was approximately 30% less in LIV (p=0.27) and, when disease was evident, involved fewer organ systems (p=0.09). Marrow-derived mesenchymal stem cells (MSC) were 52% lower (p<0.01) in LIV, and 31% lower (p=0.08) in mice lacking pathology, suggesting higher MSC levels in this model of cancer susceptibility may have contributed to tumor progression. These experiments indicate that LIV helps protect bone mass in mice inherently susceptible to cancer without compromising life expectancy, perhaps through mechanical control of stem cell fate. Further, these data reflect the numerous system-level benefits of exercise in general, and mechanical signals in particular, in the preservation of bone density and the suppression of cancer progression.

Altered composition of bone as triggered by irradiation facilitates the rapid erosion of the matrix by both cellular and physicochemical processes.

Radiation rapidly undermines trabecular architecture, a destructive process which proceeds despite a devastated cell population. In addition to the ‘biologically orchestrated’ resorption of the matrix by osteoclasts, physicochemical processes enabled by a damaged matrix may contribute to the rapid erosion of bone quality. 8w male C57BL/6 mice exposed to 5 Gy of Cs137 γ-irradiation were compared to age-matched control at 2d, 10d, or 8w following exposure. By 10d, irradiation had led to significant loss of trabecular bone volume fraction. Assessed by reflection-based Fourier transform infrared imaging (FTIRI), chemical composition of the irradiated matrix indicated that mineralization had diminished at 2d by −4.3±4.8%, and at 10d by −5.8±3.2%. These data suggest that irradiation facilitates the dissolution of the matrix through a change in the material itself, a conclusion supported by a 13.7±4.5% increase in the elastic modulus as measured by nanoindentation. The decline in viable cells within the marrow of irradiated mice at 2d implies that the immediate collapse of bone quality and inherent increased risk of fracture is not solely a result of an overly-active biologic process, but one fostered by alterations in the material matrix that predisposes the material to erosion.

Development of diet-induced fatty liver disease in the aging mouse is suppressed by brief daily exposure to low-magnitude mechanical signals

The age-induced decline in the bodys ability to fight disease is exacerbated by obesity and metabolic disease. Using a mouse model of diet-induced obesity, the combined challenge of a high-fat diet and age on liver morphology and biochemistry was characterized, while evaluating the potential of 15 min per day of high frequency (90 Hz), extremely low-magnitude (0.2 G) mechanical signals (LMMS) to suppress lipid accumulation in the liver. Following a 36-week protocol (animals 43 weeks of age), suppression of hepatomegaly and steatosis was reflected by a 29% lower liver mass in LMMS animals as compared with controls. Average triglyceride content was 101.7±19.4 μg mg−1 tissue in the livers of high-fat diet control (HFD) animals, whereas HFD+LMMS animals realized a 27% reduction to 73.8±22.8 μg mg−1 tissue. In HFD+LMMS animals, liver free fatty acids were also reduced to 0.026±0.009 μEq mg−1 tissue from 0.035±0.005 μEq mg−1 tissue in HFD. Moderate to severe micro- and macrovesicular steatosis in HFD was contrasted to a 49% reduction in area covered by the vacuoles of at least 15 μm2 in size in HFD+LMMS animals. These data provide preliminary evidence of the ability of LMMS to attenuate the progression of fatty liver disease, most likely achieved indirectly by suppressing adipogenesis and thus the total adipose burden through life, thereby reducing a downstream challenge to liver morphology and function.

Low intensity vibration mitigates tumor progression and protects bone quantity and quality in a murine model of myeloma.

Myeloma facilitates destruction of bone and marrow. Since physical activity encourages musculoskeletal preservation we evaluated whether low-intensity vibration (LIV), a means to deliver mechanical signals, could protect bone and marrow during myeloma progression. Immunocompromised-mice (n=25) were injected with human-myeloma cells, while 8 (AC) were saline-injected. Myeloma-injected mice (LIV; n=13) were subjected to daily-mechanical loading (15min/d; 0.3g @ 90Hz) while 12 (MM) were sham-handled. At 8w, femurs had 86% less trabecular bone volume fraction (BV/TV) in MM than in AC, yet only a 21% decrease in LIV was observed in comparison to AC, reflecting a 76% increase versus MM. Cortical BV was 21% and 15% lower in MM and LIV, respectively, than in AC; LIV showing 30% improvement over MM. Similar outcomes were observed in the axial skeleton, showing a 35% loss in MM with a 27% improved retention of bone in the L5 of LIV-treated mice as compared to MM. Transcortical-perforations in the femur from myeloma-induced osteolysis were 9× higher in MM versus AC, reduced by 57% in LIV. Serum-TRACP5b, 61% greater in MM versus AC, rose by 33% in LIV compared to AC, a 45% reduction in activity when compared to MM. Histomorphometric analyses of femoral trabecular bone demonstrated a 70% elevation in eroded surfaces of MM versus AC, while measures in LIV were 58% below those in MM. 72% of marrow in the femur of MM mice contained tumor, contrasted by a 31% lower burden in LIV. MM mice (42%) presented advanced-stage necrosis of tibial marrow while present in just 8% of LIV. Myeloma infiltration inversely correlated to measures of bone quality, while LIV slowed the systemic, myeloma-associated decline in bone quality and inhibited tumor progression through the hindlimbs.

Diminished satellite cells and elevated adipogenic gene expression in muscle as caused by ovariectomy are averted by low-magnitude mechanical signals

Age-related degeneration of the musculoskeletal system, accelerated by menopause, is further complicated by increased systemic and muscular adiposity. The purpose of this study was to identify at the molecular, cellular, and tissue levels the impact of ovariectomy on adiposity and satellite cell populations in mice and whether mechanical signals could influence any outcomes. Eight-week-old C57BL/6 mice were ovariectomized, with one half subjected to low-intensity vibration (LIV; 0.3 g/90 Hz, 15 min/day, 5 day/wk; n = 10) for 6 wk and the others sham vibrated (OVX; n = 10). Data are compared with age-matched, intact controls (AC; n = 10). In vivo μCT analysis showed that OVX mice gained 43% total (P < 0.001) and 125% visceral adiposity (P < 0.001) compared with their baseline after 6 wk, whereas LIV gained only 21% total (P = 0.01) and 70% visceral adiposity (P < 0.01). Relative to AC, expression of adipogenic genes (PPARγ, FABP4, PPARδ, and FoxO1) was upregulated in OVX muscle (P < 0.05), whereas LIV reduced these levels (P < 0.05). Adipogenic gene expression was inversely related to the percentage of total and reserve satellite cell populations in the muscle, with both declining in OVX compared with AC (-21 and -28%, respectively, P < 0.01). LIV mitigated these declines (-11 and -17%, respectively). These results provide further evidence of the negative consequences of estrogen depletion and demonstrate that mechanical signals have the potential to interrupt subsequent adipogenic gene expression and satellite cell suppression, emphasizing the importance of physical signals in protecting musculoskeletal integrity and slowing the fat phenotype.